WO1998055651A1 - Procede permettant de determiner la vitesse de deplacement d'une molecule dans une cellule vivante - Google Patents

Procede permettant de determiner la vitesse de deplacement d'une molecule dans une cellule vivante Download PDF

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WO1998055651A1
WO1998055651A1 PCT/US1998/010706 US9810706W WO9855651A1 WO 1998055651 A1 WO1998055651 A1 WO 1998055651A1 US 9810706 W US9810706 W US 9810706W WO 9855651 A1 WO9855651 A1 WO 9855651A1
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cell
test
nucleic acid
living
movement
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PCT/US1998/010706
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Joan C. Politz
David E. Wolf
Elizabeth S. Browne
Thoru Pederson
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University Of Massachusetts
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • This invention relates to cell biology and fluorescence microscopy.
  • FRAP fluorescence recovery after photobleaching
  • FRET fluorescence resonance energy transfer
  • FCS fluorescence correlation spectroscopy
  • FCS has been coupled with confocal microscopy techniques.
  • a scanning confocal laser microscope image has been used to detect two-dimensional spatial fluorescence intensity auto-correlation functions (Peterson et al . , J " . Biophys . 65:1135-1146 (1993)).
  • the invention features a fluorescence correlation spectroscopy method for determining the rate of movement of a fluorescent test molecule inside a living cell.
  • the method includes the steps of : (a) introducing the test molecule into the living cell; (b) placing the cell on a suitable support containing a suitable medium; (c) focusing a laser into a region of the cell to define a confocal volume; (d) sampling fluorescence intensity from the confocal volume at discrete time points, thereby producing a series of intensity values; (e) defining an autocorrelation function using the intensity values;
  • the test molecule can be monomeric. Alternatively, it can be polymeric, e.g., an oligonucleotide, or a protein.
  • the living cell can be any living cell. For example, it can be a mammalian cell such as a rat myoblast .
  • the test molecule can be introduced into the living cell by any suitable method, e.g., transfection, electroporation, microinjection, and placement of naked test molecules in culture medium bathing the cell. Cationic liposome-mediated transfection is a preferred method for use with cultured mammalian cells.
  • fluorescent moieties are useful in the invention, including Cy-3, fluorescein isothiocyanate, Texas red, tetramethylrhodamine isothiocyanate and 5 , 4-difluoro-4-bora-3a, 4a-diaza-s- indacene .
  • the method can be used to determine the rate of movement of the test molecule in a particular region of the living cell, for example, the nucleus.
  • the invention also includes a method for detecting hybridization of a test nucleic acid in a living test cell.
  • the method includes: (a) introducing the test nucleic acid into a living test cell; (b) determining a rate of movement of the test nucleic acid in the living test cell; (c) introducing a nonhybridizing nucleic acid into a living control cell; (d) determining a rate of movement of the nonhybridizing nucleic acid in the living control cell; and (e) comparing the rates of movement; wherein a lower rate of movement by the test nucleic acid indicates hybridization.
  • the test nucleic acid can be an antisense oligonucleotide .
  • the test nucleic acid typically has a length of 10 to 1000 nucleotides.
  • the test nucleic acid has a length of 15 to 500 nucleotides, and more preferably, 20 to 100 nucleotides.
  • a useful nonhybridizing nucleic acid is poly (A) .
  • the living test cell can be any living cell. For example, it can be a mammalian cell such as an L6 rat myoblast .
  • the rates of movement can be determined by any suitable method, including FCS and FRAP.
  • the test nucleic acid (or control nucleic acid) can be introduced into the test cell by any suitable method, including transfection, electroporation, microinjection, and placement of naked test molecules in culture medium bathing the cell.
  • Cationic liposome-mediated transfection is a preferred method for use with cultured mammalian cells.
  • living cell means any viable prokaryotic or eukaryotic cell having an intact plasma membrane.
  • rate of movement means the rate at which a molecule or nucleic acid naturally moves across a predetermined area within a cell.
  • test nucleic acid means a DNA or RNA of a known length.
  • nonhybridizing nucleic acid means a single-stranded DNA or RNA: (1) whose rate of movement in a control cell is compared against the rate of movement of a test nucleic acid in a living test cell; (2) whose length is equivalent to that of the test nucleic acid; and (3) that does not hybridize to any nucleic acid in the control cell under physiological conditions.
  • An example of a nonhybridizing nucleic acid is poly (A) .
  • Fig. 1 is a graph showing autocorrelation curves
  • oligo(dT) obtained from oligo(dT) alone in solution and after last addition of poly (A) RNA.
  • the dashed thick line (oligo(dT) alone) and solid thick line (hybridized oligo(dT)) are the best fit to each autocorrelation curve, using non-linear least squares analysis.
  • the autocorrelation function (G(t)) is on the y axis, and measurement time (msec) is on the x axis.
  • Fig. 2 is a histogram showing the extent of hybridization with increasing concentrations of poly (A) RNA. Hatched bars represent the fraction of oligo (dT) moving at 0.23 ms (unhybridized) . Solid bars represent the fraction of oligo (dT) moving at 1.9 ms (hybridized) .
  • Fig. 3 is a graph showing autocorrelation curves (thin lines) and best fit curves (obtained by using nonlinear least-squares analysis) fluorescein-labeled oligo (dT) (thick solid line) or fluorescein-labeled oligo (dA) (thick dashed line) in nuclei of L6 rat myoblasts .
  • Fig. 3 is a graph showing autocorrelation curves (thin lines) and best fit curves (obtained by using nonlinear least-squares analysis) fluorescein-labeled oligo (dT) (thick solid line) or
  • FIG. 4 is a graph showing autocorrelation curves (thin lines) and best fit curves for fluorescein-labeled oligo (dT) prehybridized with oligo (dA) before cellular uptake (thick dashed line) and fluorescein-labeled oligo (dT) not prehybridized with before cellular uptake (thick solid line) .
  • Fig. 5 is a graph showing distribution of oligo (dT) (solid line) compared with oligo (dA) (dashed line) . Percent particles is on the y axis, and diffusion time (msec) is on the x axis. This is a summary of FCS- determined oligonucleotide diffusion rates in L6 nuclei. Multiple FCS readings were performed in nuclei of cells treated with oligo (dT) or oligo (dA) . Vertical bars represent standard error of the mean.
  • Fig. 6 is a graph showing distribution of oligo (dT) (solid line) compared with prehybridized oligo (dT) (dashed line) . Percent particles is on the y axis, and diffusion time (msec) is on the x axis.
  • This is a summary of FCS-determined oligonucleotide diffusion rates in L6 nuclei . Multiple FCS readings were performed in nuclei of cells treated with oligo (dT) or prehybridized oligo (dT). Vertical bars represent standard error of the mean.
  • the present invention provides a method for measuring the rate of movement of a fluorescent test molecule inside a living cell using fluorescent correlation spectroscopy (FCS) .
  • the invention provides a method for detecting hybridization of a nucleic acid to a target molecule inside a living cell.
  • Measuring the rate at which a molecule moves in a living cell is useful in determining whether the molecule is freely mobile, completely immobilized or displays an intermediate degree of mobility in a particular region of a cell . This information is useful in determining the structure and function of particular regions of the cell . Furthermore, by comparing mobility rates of a fluorescently-labeled macromolecule in various regions of the cell, it is possible to identify the region at which that macromolecule interacts with an intracellular target, thereby becoming immobilized.
  • the invention is useful for detecting hybridization of a nucleic acid molecule inside a living cell.
  • the rate at which a nucleic acid molecule, e.g., an antisense oligonucleotide, moves inside a cell can be used to determine whether the molecule exists in a hybridized form, or in a single-stranded form.
  • a single- stranded nucleic acid molecule moves through a given region of the cell at a particular rate.
  • hybridized the same molecule displays a relatively lower rate of movement.
  • hybridization reduces the rate of movement of the nucleic acid.
  • the detection of hybridization inside a cell is useful in developing and testing therapeutic antisense oligonucleotides .
  • the method of the invention is a useful tool in developing drugs for the prevention and treatment of diseases, including cancer and AIDS.
  • Relevant considerations in the practice of this invention include: the type of living cell in which molecular movement is measured; the type of test molecule employed in the movement measurements; fluorescent labeling of test molecules; introduction of test molecules into living cells; preparation and use of the cells for measurement of molecular movement by fluorescence microscopy; and detection of hybridization of a test nucleic acid to a target nucleic acid in a living cell .
  • Living cells Cells that can be used in the invention include mammalian cells (e.g., fibroblasts, epithelial cells, endothelial cells, adipocytes, myoblasts, neurons), plant cells, bacterial cells, and fungal cells.
  • mammalian cells e.g., fibroblasts, epithelial cells, endothelial cells, adipocytes, myoblasts, neurons
  • plant cells e.g., fibroblasts, epithelial cells, endothelial cells, adipocytes, myoblasts, neurons
  • plant cells e.g., bacterial cells, and fungal cells.
  • the cells can be from a primary cell culture or from an established cell line.
  • Primary cells can be isolated from normal mammalian tissues or from tumors.
  • Tumor cells may be derived either from the outgrowth of migrating cells within a segment of tissue or from a biopsy specimen which has been isolated and maintained under cultured conditions.
  • Immortal cell lines can be prepared by multiple passages of primary cell cultures or may be obtained from commercial sources such as the American Tissue Type Culture Collection (ATTC) .
  • Examples of immortalized cells include, but are not limited to, HT1080 cells, HeLa cells (and derivatives thereof) , MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinoma cells, Raj i cells, Jurkat cells, Namalwa cells, HL-60 cells, Daudi cells, RPMI 8226 cells, U-937 cells, MDCK cells, Bowes Melanoma cells, WI-38VA13 subline 2R4 cells, and MOLT-4 cells and L6 rat myoblasts.
  • test Molecules The test molecule can be any molecule that is inherently fluorescent or labeled with a fluorochrome.
  • the molecule can be monomeric, e.g., a sugar, vitamin, or amino acid.
  • the molecule can be polymeric, e.g., a protein, polysaccharide, or nucleic acid.
  • Synthetic oligonucleotides are particularly useful as test molecules in the invention.
  • the basic principles of oligo design, synthesis and use are well known. Those basic principles apply generally to the design, synthesis and use of the biological degradation-resistant oligonucleotides of this invention.
  • an oligonucleotide used in this invention can vary considerably in length.
  • the preferred length of the oligonucleotide will depend on considerations such as target cell type, method of oligonucleotide introduction into the target cell, oligonucleotide concentration used, target nucleic acid type (e.g., mRNA, double- stranded DNA) , target nucleic acid length, target nucleic acid copy number, target nucleic acid G-C content, and target cell temperature.
  • target nucleic acid type e.g., mRNA, double- stranded DNA
  • target nucleic acid length e.g., mRNA, double- stranded DNA
  • target nucleic acid length e.g., target nucleic acid length
  • target nucleic acid copy number e.g., target nucleic acid G-C content
  • target cell temperature e.g., a target cell temperature
  • the length of the oligo used in this invention is in the
  • Oligonucleotide test molecules used in this invention can have any nucleotide sequence.
  • the nucleotide sequence is an antisense sequence complementary to a target nucleic acid.
  • the nucleotide sequence will depend on the sequence of the target nucleic acid.
  • the nucleotide sequence must have sufficient complementarity to the target nucleic acid to allow hybridization with the target nucleic acid, under conditions inside the target cell .
  • base pair matching between the oligo and target nucleic acid is at least 80%. More preferably, the base pair matching is approximately 100%.
  • oligonucleotide synthesis For a general discussion of oligonucleotide synthesis, see Caruthers, "Synthesis of Oligonucleotides and Oligonucleotide Analogs," in Topics in Molecular and Structural Biology, Vol . 12 : Oligodeoxynucleotides (Cohen, ed.), MacMillan Press, London, pp. 9-24. Apparatuses for automated DNA synthesis are commercially available. Preferably automated DNA synthesis is employed in obtaining oligonucleotides used in the method of this invention.
  • An oligonucleotide useful in this invention can be obtained in a two-step process.
  • the first step is synthesis of an oligonucleotide which comprises a modified base at each position in the nucleotide sequence where a fluorochrome is desired.
  • the second step is covalent attachment of fluorochromes to the modified base .
  • the purpose of the modified base used in the first step is to provide a functional group through which the fluorochrome is covalently attached to the oligo, in the second step.
  • the functional group provided by the modified base is a primary amino group.
  • the functional group is at the end of a spacer arm.
  • the functional group provided by the modified base typically bears a protecting (blocking) group, e.g., a trifluoroacetamide group.
  • a protecting (blocking) group e.g., a trifluoroacetamide group.
  • the protecting group must be removed by a suitable chemical reaction before the functional group can be used for attachment of the fluorochrome.
  • a particularly preferred modified base is a thymine analog.
  • the thymine analog can be conveniently incorporated into an oligo by means of a dT analog.
  • the dT analog is available commercially as "Amino-Modifier C6 dT" (Glen Research, Sterling, VA) .
  • Amino-Modifier C6 dT is designed for use in conventional automated D ⁇ A synthesis.
  • the trifluoroacetamide group on "Amino-Modifier C6 dT” is a protecting group. It is removed by hydrolysis during deprotection, to expose a primary amine group for use in attachment of a fluorochrome.
  • the total number, and the spacing, of the modified bases (and covalently attached protection moieties) in the oligo can vary.
  • a modified base is incorporated within five bases from the 3' end of the oligo. More preferably, a modified base is incorporated in the ultimate or penultimate base position, at the 3' end of the oligo.
  • a modified base near the 3' end of the oligo and at approximately every tenth base position in the nucleotide sequence of the oligo. Incorporation of modified bases, and thus fluorochrome moieties, closer than every ten bases causes quenching of fluorescence and concomitant loss of visual signal strength.
  • Examples of functional groups include: activated ester: amines or anilines; acyl azide: amines or anilines; acyl halide : amines , anilines, alcohols or phenols; acyl nitrile : alcohols or phenols; aldehyde : amines or anilines; alkyl halide : amines , anilines, alcohols, phenols or thiols; alkyl sulfonate : thiols, alcohols or phenols; anhydride : alcohols , phenols, amines or anilines; aryl halide : thiols ; aziridine : thiols or thioethers; carboxylic acid: amines, anilines, alcohols or alkyl halides; diazoalkane : carboxylic acids; epoxide : thiols ; haloacetamide : thiols ; hal
  • flurochromes can be covalently attached to the modified base of the oligonuicleotides .
  • flurochromes useful in this invention are
  • FITC (Molecular Probes, Inc., Eugene, OR), Texas red (Molecular Probes, Inc., Eugene, OR), Cy-3 (Biological Detection Systems, Pittsburgh, PA) , TRITC, AMCA, biotin, and digoxyigenin.
  • FITC and Texas red comprise an isothiocyanate or sulfonylchloride functional group, respectively, which reacts with primary amines.
  • FITC or Texas red is allowed to react with the primary amino group of a modified base.
  • fluorochromes onto amino groups see Agrawal et al . (1986) Nucleic Acids Res . 14:6227- 6245.
  • a useful protocol for covalent attachment of FITC or Texas red to the primary amino group of a modified base is as follows:
  • Test molecules can be introduced into test cells directly or indirectly. Direct introduction is the introduction of a previously-synthesized molecule. In indirect introduction, the test molecule is produced by biosynthesis, inside a test cell. Indirect introduction can be done by means of an expression vector encoding a protein. Unless the encoded protein (test molecule) is an inherently fluorescent protein, it must be a fusion protein. Useful fluorescent fusion moieties can be derived from green fluorescent protein, e.g., (GFP) or blue fluorescent protein (BFP) . Test molecules can be introduced directly into living cells with or without an enhancing component or condition. An example of introduction without an enhancing component or condition is the passive uptake by the cell of a naked molecule, e.g., an oligonucleotide, from a culture medium bathing the cell.
  • GFP green fluorescent protein
  • BFP blue fluorescent protein
  • Enhancing components are chemical or physico- chemical agents that promote the uptake of the introduced molecule into the cell.
  • enhancing components include calcium phosphate ⁇ see, e . g. , Sambrook et al . , Molecular Cloning - A Laboratory Manual (2d Ed.), Cold Spring Harbor Laboratory Press (1989), at pages 16.32- 16.40); DEAE-dextran (see, e . g. , Sambrook et al . , supra , at pages 16.41-16.46) and colloidal dispersion systems.
  • Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposome systems are colloidal systems useful in this invention.
  • Liposomes are artificial membrane vesicles useful for in vivo delivery. Large unilamellar vesicles can encapsulate large macromolecules. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see, e.g., Fraley, et al . , Trends Bioche . Sci . 6:77 (1981)).
  • Liposomes can be formed from cationic lipids such as N-[I-(2, 3 dioleyloxy) -propyl] -N, N, N- trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB) .
  • DOTMA N-[I-(2, 3 dioleyloxy) -propyl] -N, N, N- trimethylammonium chloride
  • DDAB dimethyl dioctadecylammonium bromid
  • liposomes are commercially available, for example, LIPOFECTIN-TM and LIPOFECTACE-TM (Gibco BRL, Grand Island, New York) , and PerfectTM (Invitrogen, San Diego, California) .
  • Methods for making liposomes are known in the art. See, e.g., Gregoriadis, Trends in Biotechnology, 3:235-241 (1985); Feigner et al . , J “ . Biol . Chem . , 269:2550-2561 (1994).
  • molecules can be introduced into the cell by the use of enhancing conditions. Examples of enhancing conditions are microinjection ⁇ see, e . g. , Leonetti et al . (1991) Proc .
  • the molecule optionally can be delivered to the living cell by administering the molecule to a host in vivo .
  • a test molecule can be targeted to a particular tissue by coupling the test molecule to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • the tissue then can be isolated and prepared by conventional methods.
  • the preferred method for introducing a fluorescent molecule into a living cell will depend on various factors, including the type of cell e.g., animal, plant or bacterial . Selection of methods suitable for introducing the fluorescent molecule into the living cell of a particular type is within ordinary skill in the art.
  • the test molecule can be introduced into a region of interest within the living cell, e.g., the cytoplasm, the nucleus, or an organelle.
  • the cytoplasm is the portion of the cell bounded by the plasma membrane other than the organelles and nucleus.
  • Prokaryotic cells and some eukaryotic cells, such as the mammalian red blood cell do not have a nucleus, and have few organelles.
  • the cytoplasm in the these cells is the entire region of the cell bounded by the plasma membrane other than any organelles that are present.
  • the nucleus is the portion of the cell bounded by the nuclear envelope.
  • organelles are mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi vesicles, lysosomes, multivesiculate bodies, peroxisomes, chloroplasts, and vacuoles.
  • Living cells into which test molecules have been introduced are transferred to a microscope stage.
  • a temperature-controlled stage can be used to maintain the cells at a suitable temperature.
  • the composition of the medium in which the cells are viewed and a the temperature at which they are viewed under the microscope will depend on the type of cell being viewed, and can be readily determined by one of ordinary skill in the art.
  • the microscope can be chambered in 5% C0 2 to ensure the maintenance of cell viability.
  • a suitable medium which maintains cell viability in a non-C0 2 -equilibrated environment can be used.
  • An example of such a medium is Liebvitz's L-15
  • FCS Fluorescence Correlation Spectroscopy
  • FCS is known in the art. Complete FCS systems, or components for assembly of a complete system, are commercially available.
  • An FCS system for use in this invention can include the following components: (a) an argon ion laser for illumination; (b) an electro-optical feedback device to minimize intensity fluctuations in the laser beam; (c) a spatial filter for rejecting high spatial frequency noise; (d) a photomultiplier to detect fluorescence and process the signal electronically and (e) computer to calculate the autocorrelation function.
  • the operational linkage of the basic components is within ordinary skill in the art.
  • FCS system is a ConfoCor fluorescence correlation spectrometer available from Carl Zeiss Jena GmbH (Oberkochen, Germany) , in combination with associated software from Evotec BioSystems GmbH (Hamburg, Germany) . An automated fluorescent microscope was interfaced to the Zeiss instrument. The equipment is also described in PCT Patent Application WO 94/16313.
  • FCS may be performed in the near field (i.e., outside the near field limit) using fiber optic illumination.
  • a confocal or conventional microscope i.e., a device that provides optical magnification, also may be used.
  • a suitable device for making this measurement allows for detection of emitted light from labeled macromolecules within a cell, with a suitable size for the portion of the region of the cell and a suitable sampling rate.
  • FCS measurements mechanical adjustments of the objective lens and the stage are done so that the cell can be observed through the oculars.
  • the desired confocal volume is established by focusing up and down over a total of approximately 10 microns from the top of the cell down through to its attachment to the plastic/glass surface. This is visualized for example, by bringing the basal surface of lamellipodia into focus.
  • the laser beam is then directed to the established region of the cell and, if necessary, refocused. The focus is once again confirmed with bright field illumination.
  • the confocal volume is established by making an FCS measurement of a known fluorescent molecule (such as FITC) in solution and setting this structural parameter in the computational algorithm.
  • a known fluorescent molecule such as FITC
  • Methods for making such measurements are known, see e.g., Widengren, (thesis) Fluorescence Correla tion Spectroscopy, Photophysical Aspects and Appli ca tions , Department of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden (1996) .
  • Fluctuations in fluorescent light intensity emitted from the test molecule can be measured over discrete time intervals.
  • the fluctuations in intensity of the fluorescent light within the confocal volume indicate the rate of movement of a molecule within that region of the living cell. For example, as the test molecule moves out of the confocal volume, the intensity of the fluorescent light decreases. As the test molecule moves into the confocal volume, the intensity of the measured light increases.
  • FRAP is known in the art.
  • FRAP For a general discussion of FRAP, see, e.g., Wolf, Methods in Cell
  • a fluorescently labeled molecule is introduced into a biological system, for example, white blood cells, sperm cells or cells grown in culture. After a period of equilibration, a portion of the cell containing fluorescent molecules is subjected to a local laser irradiation, which photochemically destroys the fluorescent groups attached to the molecules of interest . When observed by fluorescence microscopy, the photobleached area appears as a non- fluorescent spot or zone, surrounded by still fluorescent molecules. As a function of time, fluorescent molecules move into the photobleached zone. From the kinetics of this process, an apparent diffusion coefficient can be determined.
  • the invention includes a method for detecting hybridization of a test nucleic acid to a target nucleic acid inside a living cell.
  • the test nucleic acid is a DNA or RNA molecule of known length.
  • the length of the test nucleic acid is from 10 to 1000 nucleotide.
  • it is from 15 to 500 nucleotide.
  • the test nucleic acid has a length of 20 to 100 nucleotide.
  • a non-hybridizing nucleic acid is a nucleic acid which does not bind any molecule in a living cell under physiological conditions.
  • the non-hybridizing nucleic acid is a single-stranded DNA or RNA.
  • An example of a non-hybridizing oligonucleotide is poly (A) .
  • the non-hybridizing nucleic acid must be equivalent in length to the test nucleic acid.
  • the non-hybridizing nucleic acid is exposed to the same conditions as the test nucleic acid.
  • the non-hybridizing nucleic acid is introduced into the living cell using the same technique as the test nucleic acid.
  • the cell into which the non-hybridizing nucleic acid is put is the "control cell", and this cell is from the same cell population as used for the test cell.
  • the rate of movement of the test nucleic acid is compared with the rate of movement of the control nucleic acid inside the cell. A slower rate of movement of the test nucleic acid as compared with that of the control nucleic acid, is indicative of hybridization of the test nucleic acid to a cellular target .
  • a preferred test nucleic acid is an antisense oligonucleotide.
  • the antisense oligonucleotide can be an oligodeoxyribonucleotide or oligoribonucleotide (or modified form such as a phosphorothioate) that hybridizes to a nucleic acid in vivo, thereby selectively inhibiting gene expression.
  • the length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.
  • Antisense oligonucleotides can be designed to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.
  • the invention is useful for assaying the hybridization of a particular antisense oligonucleotide inside a particular type of cell.
  • the particular sequence of the oligonucleotide used according to the methods of the invention depends on the target.
  • the test oligonucleotide is a putative therapeutic oligonucleotide.
  • Combinatorial libraries of oligonucleotides may be utilized to identify and isolate novel and known oligonucleotides.
  • Each of these oligonucleotides is a putative therapeutic oligonucleotide which may be tested for therapeutic activity and may be studied according to the methods of the invention.
  • the therapeutic targets for such oligonucleotides may be nucleic acids, proteins or other cellular or foreign molecules.
  • Oligonucleotide preparation Oligo (dT) and oligo (dA) 43mers containing a 5'- aminohexyl -modified thymidine (Glen Research, Sterling, VA) at nucleotide positions 2, 12, 22, 32, 42 were synthesized using standard phosphoramidite chemistry and coupled with fluorescein isothiocyanate (Kislauskis et al . , J " . Cell Biol . , 123:165-172, 1993).
  • L6 rat myoblasts were cultured in Dulbecco- Modified Minimal Essential Medium (DMEM) with 10% heat- inactivated fetal calf serum ("serum").
  • DMEM Dulbecco- Modified Minimal Essential Medium
  • serum heat- inactivated fetal calf serum
  • For FCS about 12,500 cells in 500 ⁇ l DMEM/10% serum were plated per well of a small 8 -chambered glass-bottomed dish (Nunc, Inc., Naperville, IL) . The cells were allowed to grow for approximately 24 hours, at 37°C, in 5% C0 2 .
  • FRAP about 25,000 cells were plated onto 12 mm round glass coverslips and grown overnight as described above. All data reported were obtained with cells actively growing at subconfluence .
  • FCS measurements were made using a Zeiss-Evotec Confocor Spectrofluorimeter (Carl Zeiss Jena GmbH, Germany) in combination with compatible, commercial software (Evotec BioSystems GmbH, Germany) .
  • Zeiss-Evotec Confocor Spectrofluorimeter Carl Zeiss Jena GmbH, Germany
  • compatible, commercial software Evotec BioSystems GmbH, Germany
  • Maiti et al . Proc . Natl . Acad . Sci . USA 94:11753-11757, 1997.
  • the laser beam was focused to an exp(-2) radius of ⁇ approximately 0.2 ⁇ m in the xy plane perpendicular to the optic axis and an exp ( - 2) radius of ⁇ along the optic axis of approximately 0.6 ⁇ m.
  • the illumination source was an argon laser (488 nm and 514 nm) coupled with a fiber optic to a Zeiss Axiovert microscope.
  • the microscope had a electronic xyz stage and an electronically adjustable and positionable aperature at the image plane.
  • the detector was a fast quenching avalanche photodiode with a dead time of 33 nsec .
  • the system also had a CCD camera used for adjustment, calibration, and location of the appropriate region of the sample. Data were processed by a 288 channel logarithmic autocorrelator with adjustable sampling times from 200 nsec to 3438.8 sec.
  • the coverglass chambers with attached cells containing fluorescence-labelled oligonucleotides were placed on the microscope stage and the region of interest within the cell or the nucleus was established by mechanical adjustment of the objective and stage and observation of the cell through the oculars.
  • the desired confocal volume was established by focusing up and down over a total of approximately 10 microns from the top of the cell down through to its attachment to the plastic/glass surface. This was visualized for example, by bringing the basal surface of lamellipodia into focus.
  • the laser beam was then directed to the established region of the cell and, if necessary, refocused. The focus was once again confirmed with bright field illumination.
  • the confocal volume was established by making a FCS measurement of a known fluorescent molecule (e.g., FITC) in solution and setting this structural parameter in the computational algorithm. Methods for making such measurements are known in the art. See, e.g., Widengren, (thesis) Fluorescence Correlation Spectroscopy, Photophysical Aspects and Applica tions , Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Sweden (1996).
  • FITC fluorescent molecule
  • each experimental point in time was expressed as the product of an integer (either m or n) and ⁇ t, where m and n were time bin numbers.
  • the autocorrelation function G (m) was determined.
  • ⁇ , ⁇ 2 /4D i
  • 200 ⁇ l of 20 nM oligonucleotide in water (or in Leibovitz L-15 medium) were placed in one well of an 8-chambered dish and the laser beam was focused 150 ⁇ m above the dish bottom in a known confocal volume of approximately 1 x 10 "15 liter.
  • This medium supported continued cell viability on the microscope stage at room temperature without C0 2 and also contributed only low background autofluorescence signal to FCS readings.
  • the laser beam was then focused (again in the same known confocal volume) inside the nucleus of a treated (or untreated) cell by adjusting both the stage position and focal plane.
  • Neutral density filters were used to attenuate laser intensity and reduce detection of cellular autofluorescence .
  • the fluorescence intensity was recorded, and the z plane was adjusted up or down 1-2 ⁇ m to obtain the maximal intensity reading. Care was taken to focus inside the nucleus and to avoid nucleoli. In these myoblasts, the nucleus was about 5-8 ⁇ m thick.
  • the optimal fit was chosen by comparing the following parameters among possible fits: shape of residual curve, value of relative least square, and standard deviations associated with computed fractions and diffusion times. To combine and compare data among different experiments in cells, the best fits from all the measurements from each experiment were sorted into five distinct ranges of diffusion times. The average fraction of oligo and its average diffusion time within each of the five ranges was then calculated after weighting each value to the number of fluorescent particles present in each range .
  • a 7.5 kb poly (A) RNA was purchased from Gibco BRL,
  • FCS FCS to monitor hybridization of oligo (dT) to poly (A) RNA, and to measure the rate of movement of the oligo (dT) /poly (A) RNA hybrid in aqueous solution.
  • This curve best fit a two-component model, with one species fixed at the free oligo diffusion time (0.23 msec) , and a slower component moving at a diffusion time of 1.9 msec.
  • D 1.0 x 10 "7 cm 2 /sec) .
  • Oligo (dA) diffusion time was not altered upon addition of poly (A) RNA, confirming that the observed binding of oligo (dT) to poly (A) RNA represented specific hybridization.
  • oligo (dT) 43 The intracellular mobility of fluorescently- labeled oligonucleotides oligo (dT) 43 , and oligo (dA) 43 , delivered into cultured rat L6 myoblasts was investigated by FCS.
  • FCS The mean translation diffusion times of the oligonucleotides in the nucleus were measured.
  • rate of movement of these two oligonucleotides was measured in aqueous solution.
  • the oligo (dA) 43 i.e., nonhybridizing oligonucleotide, displayed movemant rates within the same order of magnitude as it rate of movement in the aqueous solution.
  • Oligo (dA) 43 (solution) 0.95 ⁇ 0.11
  • the initial findings on rate of molecular movement were confirmed and expanded in subsequent experiments.
  • Cells were grown in medium containing fluorescein-labeled oligo (dT) or oligo (dA) for 2 hours, followed by incubation for at least 1 hour in oligonucleotide-free medium, to allow efflux of excess unbound oligonucleotide .
  • oligo (dT) The diffusion times of the oligos within the nucleus were then measured by FCS. As shown in Fig. 3, the shapes of the autocorrelation functions were different for oligo (dT) vs. oligo (dA). A substantial fraction of the total oligo (dT) moved at a slower rate than oligo (dA) . This was indicated by the tail of the correlation curve for oligo (dT) extending out to slower diffusion times.
  • oligo (dT) fluorescein-labeled oligo (dT) was prehybridized in vi tro with a 1.5 molar excess of unlabeled oligo (dA) before incubation with cells.
  • This prehybridization step changed the shape of the autocorrelation curve to show a reduction in the amount of oligo (dT) moving at the slower rate in the nucleus, as would be expected if the oligo (dT) already bound to oligo (dA) were unable to hybridize to the intranuclear poly(A) RNA targets (Fig. 4).
  • the y intercept of the autocorrelation curves is the inverse of the number of fluorescent particles in the confocal volume.
  • the slow-moving molecules present in oligo (dT) treated cells with diffusion times of 1 msec, or more (diffusion coefficients between 1 x 10 "10 and 9 x 10 "8 cm 2 /sec) , represented an average of 45% of the total oligo (dT) molecules detectable in the nucleus by FCS (Table 3, columns 3-5; Figs. 5 and 6) .
  • This fraction likely contained oligo (dT) hybridized to poly (A) RNA, because we saw much less of this fraction in cells treated with oligo (dA) (10%), or in cells treated with oligo (dT) that had been prehybridized in vi tro to oligo(dA) (22%) (Table 3, Figs. 5 and 6, respectively).
  • the concentration of rhodamine-conjugated dextran in 150 ⁇ l of cell culture medium was 0.3 ⁇ M, and this volume of
  • 150 ⁇ l was mixed with 150 ⁇ l of cell culture medium containing 1.5 ⁇ l of PFX-5, and the resulting 300 ⁇ l mixture was then added to the chamber of cells (having first r removed the prior medium) .
  • the intracellular mobility of fluorescently labeled dextran delivered into cultured rat myoblasts also has been investigated by fluorescence correlation spectroscopy.
  • the mean translation diffusion times of the labeled dextran in the nucleus were measured. Parallel measurements were made in aqueous solution.
  • the dextran was found to display molecular mobility within the same order of magnitude as the diffusion rate of the dextran measured in an aqueous solution. Results of the study are presented in Table 4 is the diffusion coefficient .
  • FRAP measurements were performed using a previously described workstation (Wolf, Methods in Cell Biol, 30:271-306, 1989). All FRAP measurements were made at room temperature with a Zeiss 63 X oil 1.4 NA plan Apochromat objective. Solution measurements were performed using 0.05 mm square capillary tubes (Vitrodynamics) containing 20 nM fluorescein-labeled oligo (dT) or oligo (dA) in Leibovitz L-15 medium. FRAP measurements on nuclei were performed on cells growing in Leibovitz L-15 (with serum), on coverslips mounted on slides.
  • Photobleaching was at -1.3 mW for 10 msec with an exp(-2) beam radius of 1 ⁇ m (-40 kW/cm 2 ) . Monitoring intensity was -0.13 ⁇ W.
  • Measured diffusion coefficients were normalized using the published diffusion coefficient for BSA (see Lang et al . , J. Cell Biol, 102:1183-1190, 1986) to correct for changes in beam radius along the optical axis. Because our FRAP instrument had a beam radius approximately four times larger than our FCS instrument, we had to increase the amount of fluorescent oligo signal in cells in order to carry out FRAP measurements. To do this, L6 myoblasts were incubated with oligo in the presence of a cationic lipid.
  • oligo (dT) populations were found to move with a diffusion coefficient of 1.2 x 10 7 cm 2 /sec (Table 5), a rate intermediate between the fast and slower rates observed for oligo (dT) using FCS (see Table 3) . This is consistent with the fact that our FRAP instrument calculates one overall average rate of diffusion back into the bleached area.
  • the FRAP- determined diffusion coefficient for oligo (dT) corresponded well to the average FCS diffusion coefficient of 8.7 x 10 "8 cm 2 /sec calculated from FCS data treated as a one component model (Table 5) .
  • FCS data treated as a one component model Table 5
  • FRAP was used to measure diffusion in oligo (dA) treated cells, an average diffusion coefficient of 2.6 x 10 "7 cm 2 /sec was obtained (Table 5) , reflecting the higher percentage of oligo (dA) as free oligo.
  • FCS-measured diffusion rates in the nucleus of living cells are well within the range of values obtained with FRAP.
  • FRAP recovery rates were 80 ⁇ 3%. Errors are standard errors of the mean.

Abstract

Cette invention concerne un procédé permettant de déterminer la vitesse de déplacement d'une molécule dans une cellule vivante, ceci en effectuant une spectroscopie de corrélation de fluorescence (SCF). Cette invention concerne également un procédé permettant de détecter l'hybridation d'un acide nucléique dans une cellule vivante. La figure représente un graphique qui montre les courbes d'auto-corrélation ainsi que les courbes d'ajustement optimal pour des oligonucléotides marqués par fluorescence dans des noyaux de myoblastes L6 chez le rat.
PCT/US1998/010706 1997-06-05 1998-05-26 Procede permettant de determiner la vitesse de deplacement d'une molecule dans une cellule vivante WO1998055651A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1120653A1 (fr) * 1999-07-23 2001-08-01 Olympus Optical Co., Ltd. Procede visant a examiner une substance ayant une interaction avec un recepteur hormonal
WO2010083852A1 (fr) * 2009-01-26 2010-07-29 Tethis S.R.L. Dispositif microfluidique fonctionnalisé pour immunofluorescence
CN112781958A (zh) * 2021-02-02 2021-05-11 江南大学 一种表征乳液分子特定成分流动性的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BERLAND K. M., ET AL.: "TWO-PHOTON FLUORESCENCE CORRELATION SPECTROSCOPY: METHOD AND APPLICATION TO THE INTRACELLULAR ENVIRONMENT.", BIOPHYSICAL JOURNAL, CELL PRESS, US, vol. 68., 1 February 1995 (1995-02-01), US, pages 694 - 701., XP002911901, ISSN: 0006-3495 *
POLITZ J. C., ET AL.: "CHARACTERIZATION OF HYBRIDIZATION BETWEEN SYNTHETIC OLIGODEOXYNUCLEOTIDES AND RNA IN LIVING CELLS.", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD., GB, vol. 23., no. 24., 1 January 1995 (1995-01-01), GB, pages 4946 - 4953., XP002911900, ISSN: 0305-1048 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1120653A1 (fr) * 1999-07-23 2001-08-01 Olympus Optical Co., Ltd. Procede visant a examiner une substance ayant une interaction avec un recepteur hormonal
EP1120653A4 (fr) * 1999-07-23 2003-05-21 Olympus Optical Co Procede visant a examiner une substance ayant une interaction avec un recepteur hormonal
WO2010083852A1 (fr) * 2009-01-26 2010-07-29 Tethis S.R.L. Dispositif microfluidique fonctionnalisé pour immunofluorescence
CN112781958A (zh) * 2021-02-02 2021-05-11 江南大学 一种表征乳液分子特定成分流动性的方法

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